Large protein machines control almost all biological processes, including DNA replication, RNA processing, and both protein synthesis and degradation. The 26S proteasome is responsible for the great majority of protein degradation in eukaryotes and is essential for numerous cellular pathways. Aberrant proteasomal activity impacts many human diseases, such as neurodegenerative disorders, cancer, and diabetes. Like many multisubunit catalytic complexes, the proteasome forms ring-shaped structures. The proteasome consists of a cylindrical 20S proteasome core particle (CP) with a 19S regulatory particle (RP) on one or both ends. In the CP, two outer heteroheptameric ? rings sandwich a pair of ? rings; the latter creates the central proteolytic chamber. Each RP can be divided into ld and base complexes. The RP base includes six ATPases that form a heterohexameric ring; the RP binds and unfolds substrates and drives them into the CP interior. How these large, essential complexes are assembled in vivo remains poorly understood. Proteasome assembly appears to be an ordered process conserved across species. As proteasomes are highly abundant, assembly must occur with high fidelity to avoid excessive accumulation of nonproductive and potentially toxic off-pathway intermediates. Both CP and RP assembly depend on dedicated assembly chaperones. The long-range goal of this grant is to delineate the pathways of eukaryotic 26S proteasome biogenesis. The proteasome has emerged as an important target for anti-cancer treatment and other therapies. Interfering with its assembly will provide a new approach for pharmaceutical intervention. The proposed experiments use a combination of genetic, biochemical, cell biological and biophysical methods and are focused primarily, but not entirely, on the model eukaryote Saccharomyces cerevisiae, which has a 26S proteasome very similar to the human complex. The first major goal will be to decipher the assembly-promoting mechanism of a putative novel CP assembly factor and how it interacts functionally with known CP assembly chaperones. A second goal is to determine how CPs with alternative ?-subunit arrangements assembles in both yeast and human cells and to evaluate their functional significance. In a third aim, the focus is on a set of RP assembly chaperones (RACs) to determine their contributions to proper assembly of the ATPase ring of the RP base; the mechanism of RP lid-base joining will also be probed. Lastly, experiments will address how proteasome assembly is linked to import into the nucleus and how proteasomes in quiescent cells rapidly relocalize from cytoplasmic foci to the nucleus in response to glucose addition.